We have begun a new approach to vaccines. We directly harness the properties of dendrific cells (DCs) to improve vaccine efficacy, with emphasis on T cell mediated immunity. Previously we designed protein vaccines to improve delivery of HIV gag to DCs in mice. Directed or targeted delivery of gag within a monoclonal antibodies to the DEC-205 receptor on DCs gave a large increase in immunogenicity relative to nontargeted protein as well as protection in a mouse model. A newer example of DC targeting involves DNA vaccines, as will be studied here. The vaccine encodes a fusion protein comprised of a single chain Fv antibody to DCs fused to HIV gag. Immunizing and protective efficacy increased ~100 fold. We have 4 aims to study mechanisms and improve DNA vaccines along with several key collaborators: 1) Delivery of DNA vaccine anfigens to DCs is currently monitored indirectly, by virtue ofthe immunity that is induced. To gain direct data on DC uptake and processing of vaccines in mice, we will a) tag vaccine proteins with a sensitive system we developed called OLLAS; b) use antibodies to MHC-peptide, and c) isolate MHC products from targeted DCs to identify the presented pepfides. 2) With these assays we will then compare different DC receptors for targeting vaccine protein to these critical presenting cells. We have prepared hybridomas and cloned the heavy and light chains to additional DC receptors like Langerin/CD207 and DC-SIGN/CD209, so that we can prepare single chain Fv gag fusion DNA vaccines to other DC targets. 3) To improve DNA vaccine efficacy at the level of DCs, we will study the immunostimulatory effects of different DC maturation stimuli, including Ig C region sequences that preferenfially ligate activating FcyR. 4) To extend DC-based DNA vaccines to the SIV macaque model, we already have prepared anti-human DEC-205 antibodies that react with monkey DEC-205 and mediate efficient cross presentation of gag on MHC class I, and we have prepared human DEC-205 transgenic mice to ensure that anti-rhesus/human single chain DEC successfully targets and presents SIV antigens in vivo. This will allow us to determine if single chain Fv gag fusion DNA vaccines successfully target to DCs in monkey lymphoid tissues.